Characterising Planktonic Dinoflagellate Diversity in Singapore Using DNA Metabarcoding
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Metabarcoding and Metagenomics 2: 1–14 DOI 10.3897/mbmg.2.25136 Research Article Characterising planktonic dinoflagellate diversity in Singapore using DNA metabarcoding Yue Sze1, Lilibeth N. Miranda2, Tsai Min Sin2,†, Danwei Huang1,2 1 Department of Biological Sciences, National University of Singapore, Singapore 117558, Singapore. 2 Tropical Marine Science Institute, National University of Singapore, Singapore 119227, Singapore. † Deceased. Corresponding author: Danwei Huang ([email protected]) Academic editor: Thorsten Stoeck | Received 19 March 2018 | Accepted 24 April 2018 | Published 17 May 2018 Abstract Dinoflagellates are traditionally identified morphologically using microscopy, which is a time-consuming and labour-intensive process. Hence, we explored DNA metabarcoding using high-throughput sequencing as a more efficient way to study planktonic dinoflagellate diversity in Singapore’s waters. From 29 minimally pre-sorted water samples collected at four locations in western Singapore, DNA was extracted, amplified and sequenced for a 313-bp fragment of the V4–V5 region in the 18S ribosomal RNA gene. Two sequencing runs generated 2,847,170 assembled paired-end reads, corresponding to 573,176 unique sequences. Sequenc- es were clustered at 97% similarity and analysed with stringent thresholds (≥150 bp, ≥20 reads, ≥95% match to dinoflagellates), recovering 28 dinoflagellate taxa. Dinoflagellate diversity captured includes parasitic and symbiotic groups which are difficult to identify morphologically. Richness is similar between the inner and outer West Johor Strait, but variations in community structure are apparent, likely driven by environmental differences. None of the taxa detected in a recent phytoplankton bloom along the West Johor Strait have been recovered in our samples, suggesting that background communities are distinct from bloom communities. The voluminous data obtained in this study contribute baseline information for Singapore’s phytoplankton communities and prompt future research and monitoring to adopt the approach established here. Key Words high-throughput sequencing; Johor Strait; molecular operational taxonomic unit; phytoplankton; Singapore Strait Introduction selves (Taylor et al. 2008). As part of the phytoplankton community, they are primary producers that form the base Dinoflagellates (Alveolata: Dinophyceae) are a diverse of food chains and are a large constituent of the aquatic and abundant group of unicellular protists found in both system’s food web (Spector 1984; Taylor et al. 2008). marine and freshwater environments. With more than In the marine environment, there are more than 1,500 2,000 living species described (Gómez 2012), they are one species including both photosynthetic and non-photosyn- of the most functionally important organisms in a variety thetic types (Taylor et al. 2008, Gómez 2012). Massive of aquatic ecosystems (Spector 1984; Taylor et al. 2008). proliferation and accumulation of planktonic dinoflagellates On top of being species rich, they have highly diversified can lead to water colouration and certain species are also morphologies, physiologies and biochemical properties known to cause damage to marine ecosystems as harmful (Hackett et al. 2004; Taylor et al. 2008). Dinoflagellates algal blooms or ‘red tides’ (Berdalet et al. 2016). Fish kills can be armoured or unarmoured (naked), based on the and other animal mortalities are common especially when presence or absence of thecal plates respectively (Netzel the bloom involves toxin-producing dinoflagellates such as and Dürr 1984). They play diverse ecological roles—as Karenia brevis (Landsberg et al. 2009). Incidents of paralyt- autotrophs, heterotrophs or mixotrophs—and can be endo- ic shellfish poisoning by dinoflagellates such as Alexandri- symbionts of other organisms, notably the shallow-water um fundyense have serious health implications higher up the reef-building corals, or host other endosymbionts them- food chain especially where human consumption is involved Copyright Yue Sze et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Sze et al.: Characterising planktonic dinoflagellate diversity in Singapore using DNA metabarcoding (Tan and Lee 1986; John et al. 2015). For example, in 2008, simultaneously, rapidly and cost-effectively (Aylagas et al. an intense bloom in the St Lawrence Estuary was found to 2016). Important ecological applications of metabarcoding be responsible for the death of many fish, bird and mammal include diet and biodiversity analyses, with environmen- species and extremely hazardous levels of paralytic shell- tal samples of various ecosystems obtained mainly from fish toxins produced by the dinoflagellate were detected in faeces (Srivathsan et al. 2015; Guillerault et al. 2017), soil edible mussels collected during the event (Starr et al. 2017). (Andersen et al. 2012; Treonis et al. 2018) or water sam- The most common and earliest method of observing ples (Thomsen et al. 2012; Yamamoto et al. 2017). phytoplankton is through light microscopy, which is time In the marine environment, metabarcoding has been consuming, labour intensive and demands a high level useful for studying both benthic and planktonic eukary- of taxonomic expertise (Sinigalliano et al. 2009; Medlin otic diversity (Fonseca et al. 2010, 2014; Logares et al. 2013; McNamee et al. 2016). Some species, such as those 2014; de Vargas et al. 2015; Guardiola et al. 2015; Le in Alexandrium Halim, 1960, are relatively featureless Bescot et al. 2016; Tragin et al. 2018). Studies have tar- and furthermore belong to species complexes, rendering geted the 18S rRNA and successfully characterised phy- them challenging to identify morphologically (John et al. toplankton communities, which are more diverse than 2005; Anderson et al. 2012; John et al. 2014). In addition, previously thought (de Vargas et al. 2015; Le Bescot et naked dinoflagellates are also difficult to identify from al. 2016). Small and symbiotic species have escaped tra- preserved samples. Hence, more efficient approaches to ditional microscopic detection but can now be identified study dinoflagellates have been developed, including the via metabarcoding (Le Bescot et al. 2016, Leblanc et al. use of scanning electron microscopy (Jung et al. 2010), 2018). Here we use this technique to study phytoplankton natural fluorescence (Karlson et al. 2010), flow cytom- communities in the coastal waters of Singapore. etry (Sinigalliano et al. 2009), high performance liquid The earliest studies of Singapore’s marine phytoplank- chromatography (HPLC) (Gin et al. 2006) and molecular ton communities were carried out in the mid-1900s, fo- methods (Karlson et al. 2010). cusing on the Singapore Strait (Tham 1953, 1973; see Molecular techniques exploit the genetic distinction Lee et al. 2015). It has long been hypothesised that the between species to identify and quantify species and have succession of phytoplankton species is likely caused by played an integral role in our understanding of the sys- the biological history of water, stability and turbulence tematic positions and evolutionary relationships amongst of the water column and monsoon-driven currents (Tham organisms (Karlson et al. 2010). Examples of earlier mo- 1973). There have also been new species discoveries lecular methods include cloning and sequencing (Zard- (Holmes 1998) and new species records for Singapore oya et al. 1995), as well as heteroduplex mobility assay (Leong et al. 2015). More recently, studies have begun (Oldach et al. 2000). More recently, DNA barcoding has to use more advanced techniques, which include the use been successful in identifying dinoflagellates (Stern et al. of extracted chlorophyll measurements (Gin et al. 2000), 2010). It uses the amplified sequence from a short, stan- flow cytometry (Gin et al. 2003), HPLC pigment analysis dardised DNA locus as diagnostic characters for species (Gin et al. 2003, 2006), spectral fluorometric characteri- identification (Lin et al. 2009; Shokralla et al. 2012). sation (Kuwahara and Leong 2015) and also molecular Loci tested for dinoflagellates include the nuclear small methods (Tang et al. 2007; Leong et al. 2015). The con- ribosomal subunit (18S rRNA) (Lin et al. 2006), large ri- sensus of these efforts is that, amongst all phytoplank- bosomal subunit (28S rRNA; D1/D2 region) (Elferink et ton groups, diatoms have the highest abundance except al. 2017), internal transcribed spacers (Stern et al. 2012) during dinoflagellate blooms (Gin et al. 2006; Leong et and the mitochondrial cytochrome c oxidase subunit I al. 2015). Particularly in the Johor Strait, there appears and cytochrome b (Lin et al. 2009). The mitochondrial to be an inverse relationship between dinoflagellate and genes cannot be easily amplified from all dinoflagellate diatom counts (Chia et al. 1988). taxa (Stern et al. 2012) and, amongst the nuclear loci, 18S There have been more than 20 positively identified spe- rRNA remains the most well-sequenced marker in public cies of dinoflagellates reported from Singapore’s waters, databases due to their widespread use in dinoflagellate but those which are known in detail have been studied systematics (Mordret et al. 2018). mainly because they are associated with harmful algal Improved technologies for performing high-through- blooms (Leong et al.